We thank Mr. O. Rios for his technical assistance.

这项研究获得批准，由医疗机构和国家理工学院的研究和推进研究中心的地方伦理委员会使用人的材料。 1.肿瘤样品采集<ol><li>通过反式蝶骨手术过程14获得从病人垂体腺瘤组织活检。 </li><li>收集垂体腺瘤样品，将其放入含有50有10％胎儿牛血清（FBS）的富集的新鲜199培养基（M199），0.6毫克/毫升碳酸氢钠，2.4毫克/毫升HEPES和1％的混合物的无菌玻璃瓶抗生素（青霉素/链霉素）在pH 7.4。保持在玻璃瓶中在冰上的样品。 注意:肿瘤组织可以在手术后进行处理几个小时。 </li><li>运送肿瘤样品在玻璃瓶上冰的实验室。 </li></ol> 2.酶组织解离溶液的制备中，胰蛋白酶抑制剂的解决方案和解决方案海藻酸钠注意:在此之前的细胞培养过程制备以下的溶液。过滤用0.22微米的膜过滤器的所有解决方案，使用前。 <ol><li>通过在5ml M199-无血清的5毫克胶原酶（I型）的溶解制备胶原酶1％的溶液。 </li><li>准备由0.01克胰蛋白酶溶解在10ml的PBS-EDTA的0.3％胰蛋白酶0.01％的溶液。 </li><li>由2毫克的大豆胰蛋白酶抑制剂的溶解于20ml的M199-无血清的制备0.1％胰蛋​​白酶抑制剂溶液。 </li><li>通过在4毫升的胰蛋白酶抑制剂溶液中溶解20微升的DNA酶I 134 U / ml的制备DNA酶溶液。 </li><li>由22.8毫克的EGTA溶解在40毫升155毫摩尔NaCl的制备EGTA溶液（1.2M）。将pH调节至7.4。继续在50 ml的终体积。 </li><li>通过在50毫升155毫摩尔NaCl的溶解238毫克的HEPES制备的HEPES溶液（20毫摩尔）。 </li><li>通过溶解1.2准备1.2％海藻酸钠溶液克藻酸钠在100ml的HEPES溶液。高压灭菌的藻酸盐溶液。 </li><li>通过在50毫升HEPES液750毫克氯化钙溶解制备钙溶液（102毫米），调节pH值至7.4 </li></ol> 3.原代细胞培养和海藻酸钠封装<ol><li>层流柜内，将肿瘤组织中含有大约20毫升的PBS 的 Ca 2+和Mg 2+游离，pH值7.4（PBS *）的35毫米培养皿中。 </li><li>取组织用外科镊子，并将其放置在含有新鲜的PBS *另一个陪替氏培养皿，轻轻洗净组织。直到红血细胞和碎片除去重复此步骤。 </li><li>使用镊子手术，按住垂体腺瘤组织，并用小手术剪，切成小块。用巴氏吸管圆边，在RT转移组织碎片和PBS *入15ml管中，离心68 xg离心10分钟。 注意:要圆一个巴斯德的边缘吸管，微火焰的吸管尖在本生灯。 </li><li>使用巴斯德吸管除去PBS *和添加3-5毫升胶原酶溶液（大约5毫升的组织为65mm 3），通过颠倒混合的两到三倍。孵育在胶原酶的组织为恒定旋转，在37℃30分钟。 </li><li>离心管中，在68×g离心在RT 10分钟，并用巴氏吸管除去胶原酶。 </li><li>添加5毫升DNA酶，并通过反相混合的两到三倍，然后离心管在68×g离心在RT 10分钟。 注意:如果组织未完全离解，在37℃下用胰蛋白酶进行第二次酶消化3分钟。通过加入2倍体积的胰蛋白酶抑制剂溶液的停止消化。 </li><li>吸用巴氏吸管将上清液并丢弃。重悬在1体积的EGTA溶液沉淀，用2体积海藻酸钠溶液中混合。 </li><li>轻轻地混合细胞悬液和海藻酸钠通过用巴氏吸管吸取。采取与21g的针头的无菌注射器，取出活塞;使用移液管装入与藻酸盐和细胞溶液的注射器。 </li><li>轻轻插入柱塞插入注射器并仔细分配所述藻细胞溶液滴逐滴入含约30毫升富钙溶液的玻璃烧杯中。 </li><li>放置在注射器的针约5cm至氯化钙溶液。在与氯化钙接触藻细胞悬浮液的凝胶，以形成球形珠。保持藻酸盐珠在5分钟的钙溶液。 注:搅拌玻璃烧杯，其中珠被丢弃。用打圈的手慢慢移动，以防止它们粘在一起。 </li><li>小心地取出用巴氏吸管中的钙溶液中，并用3-5毫升M199与FBS的20％的富集洗珠两次。 </li><li>转移海藻酸钠珠成常规T25组织培养烧瓶使用无菌刮刀，在5％的CO 2的潮湿培养箱中添加4-5 M199毫升富含20％FBS和孵育在37℃。更换培养基每三天。 </li></ol> 4.解放从海藻细胞注意:在此之前，从藻酸盐珠解放细胞，用聚D-赖氨酸涂层13毫米直径的盖玻片。 注意:硫酸是一种高腐蚀性酸和APTS可引起眼和皮肤有刺激性。 USE乳胶手套来处理这些试剂。 <ol><li>在通风橱中，用点镊子浸泡在含有20％硫酸水溶液，在室温1小时的玻璃陪替氏培养皿的盖玻片。 </li><li>滗硫酸，并用蒸馏水洗涤盖玻片五次，每次5分钟。使用镊子点的地方干净盖玻片含0.1 M氢氧化钠5分钟，在室温15培养皿。 </li><li>倒出氢氧化钠洗一次与夹带原导致水，吸水和干燥的盖玻片。 </li><li>使用一个微量涂布的盖玻片与APTS（氨基丙基三乙氧基硅烷）处理4分钟（用500微升6盖玻片）淹没培养皿用蒸馏水。迅速吸出水，​​并继续用蒸馏水洗，移动与点镊子盖玻片以允许水底下穿过。 </li><li>吸水和干燥盖玻片，用点镊地方的盖玻片到一个干净的培养皿中，并在3次，每次45秒消毒他们的微波的内部。添加1000微升无菌水至100微克多聚-D-赖氨酸（0.1毫克/毫升）。 </li><li>孵育大约50微升聚-D-赖氨酸的盖玻片在RT 5分钟。完全吸出聚-D-赖氨酸。 注意:这是重要的，因为可溶性聚D-赖氨酸的培养基中能抑制细胞增殖。 </li><li>冲洗盖玻片用无菌水五次，每次5分钟。吸出STerile水并允许盖玻片到层流柜内干燥。 </li><li>分析肌动蛋白骨架，解放从海藻垂体细胞。用1毫升枪头，采取一些的藻酸盐珠培养瓶中，将它们放入15毫升管。 </li><li>添加5毫升55 mM柠檬酸钠（溶解323.6毫克的Na 3 C 6 H 5 O 7，2H 2 O在20ml的HEPES溶液）和离心机在68×g离心在RT 10分钟。吸出上清液并丢弃。 </li><li>重悬在1毫升温暖M199的20％FSB丰富了颗粒;轻轻地用巴斯德吸管混合细胞悬浮液。 注意:毒伞素和DAPI是有毒的。使用手套来处理这些试剂。 </li><li>计数细胞，转移125微升的0.4％台盼蓝溶液到1.5ml管中。添加M199的75微升和50微升在最后步骤（稀释倍数= 5）中得到的细胞悬浮液。调匀。 </li><li>使用微量尖PLACË约10微升的血细胞计数器室台盼蓝细胞悬液。 </li><li>使用具有相衬照明倒置显微镜计数在血球计数器中的细胞。算上1mm的中心广场和四个1毫米角区域的所有单元格。通过用稀释因子乘以每平方平均计数得到每毫升细胞密度- 10 4例如，如果每平方的平均计数为45细胞×5×104 = 2.25×106细胞/ ml。 注意:计数细胞在血细胞计数器室十正方形。不要指望细胞触及底部和右侧的中间线。 </li><li>种子35,000个细胞上覆盖有聚-D-赖氨酸的直径13mm玻璃盖玻片上。 </li></ol> 5.微丝骨架安排<ol><li>培养48小时后，取出培养基用巴氏吸管赶紧每PBS的盖玻片加入500μl，立即取出PBS。 </li><li>修复和通透Ť他细胞用每固定缓冲液（0.1M PIPES，pH值6.75，4％的PEG-6000，1mM的EGTA，1mM的MgSO 4上，0.5％的Triton X-100和2％的甲醛）的盖玻片500μl的10分钟，在37℃ C </li><li>除去固定缓冲区和添加每3.5％多聚甲醛的盖玻片500μl的在PBS中在RT下30分钟。取出多聚甲醛溶液。 </li><li>通过加入每PBS中盖玻片500微升，每次5分钟，洗涤盖玻片三次。冲洗盖玻片在室温下，在不断搅拌。 </li><li>孵育用50mM氯化铵的细胞10分钟，吸出氯化铵溶液，并重复步骤5.4。 </li><li>孵育在PBS的1％BSA（抗体，免蛋白酶-）细胞30分钟，除去BSA溶液，并重复步骤5.4。 </li><li>孵育罗丹明缀合的鬼笔环肽的50微米的细胞在室温下7分钟，重复步骤5.4。 </li><li>孵育DAPI的PBS稀释在室温下5分钟，重复步骤5.4的255微米的细胞。最后安装ŧ他与一安装平台盖玻片并用指甲油密封。 </li><li>获得的使用共聚焦激光扫描显微镜具有63X物镜的肌动蛋白细胞骨架的安排的图像，在541nm处的激发波长。 </li></ol>

<ol>
	<li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+Extracellular+Matrix+Mimics+for+3D+Cell+Culture.">Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture.</a> Biotechnol. Bioeng. 103 (4), 655-663 (2009).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65 (5), 605-610 (1999).</li><li>Andersen, T., Strand, B., Formo, K., Alsberg, E., Christensen, B., Rauter, A. P., Lindhorst, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginates+as+biomaterials+in+tissue+engineering.">Alginates as biomaterials in tissue engineering.</a> Carbohydrate chemistry. Vol. 37, 227-258 (2012).</li><li>Jonitz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+capacity+of+human+chondrocytes+embedded+in+alginate+matrix.">Differentiation capacity of human chondrocytes embedded in alginate matrix.</a> Connect. Tissue. Res. 52 (6), 503-551 (2011).</li><li>Hill, E., Boontheekul, T., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+scaffolds+to+enhance+transplanted+myoblast+survival+and+migration.">Designing scaffolds to enhance transplanted myoblast survival and migration.</a> Tissue. Eng. 12 (5), 1295-1304 (2006).</li><li>Purcell, E. K., Singh, A., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+composition+effects+on+a+neural+stem+cell-seeded+scaffold.">Alginate composition effects on a neural stem cell-seeded scaffold.</a> Tissue. Eng. Part. C. Methods. 15 (4), 541-550 (2009).</li><li>Wee, S., Gombotz, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+release+from+alginate+matrices.">Protein release from alginate matrices.</a> Adv. Drug. Deliv. Rev. 31 (4), 267-285 (1998).</li><li>Tashjian, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+strains+of+hormone-producing+pituitary+cells.">Clonal strains of hormone-producing pituitary cells.</a> Methods. Enzymol. 58, 527-535 (1979).</li><li>Fasekas, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+pituitary+adenomas+in+cell+cultures+by+light+and+electron+microscopic+morphology+and+immunolabeling.">Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling.</a> Folia. Histochem. Cytobiol. 43 (2), 81-90 (2005).</li><li>Kohler, P. O., Bridson, W. E., Rayford, P. L., Kohler, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+Production+by+Human+Pituitary+Adenomas+in+Culture.">Hormone Production by Human Pituitary Adenomas in Culture.</a> Metabolism. 18 (9), 782-788 (1969).</li><li>Kletzkky, O. A., Marrs, R. P., Rundall, T. T., Weiss, M. H., Beierle, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+and+suspension+culture+of+human+prolactin-secreting+pituitary+adenoma.">Monolayer and suspension culture of human prolactin-secreting pituitary adenoma.</a> Am. J. Obstet. Gynecol. 138 (6), 660-664 (1980).</li><li>Melmed, S., Odenheimer, D., Carlson, H. E., Hershman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+functional+human+pituitary+tumor+cell+cultures.">Establishment of functional human pituitary tumor cell cultures.</a> In Vitro. 18 (1), 35-42 (1982).</li><li>Thompson, K. W., Vincent, M. M., Jensen, F. C., Price, R. T., Schapiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hormones+by+human+anterior+pituitary+cells+in+serial+culture.">Production of hormones by human anterior pituitary cells in serial culture.</a> Proc. Soc. Exp. Biol. Med. 102 (2), 403-408 (1959).</li><li>Cappabianca, P., Cavallo, L. M., Solari, D., Stagno, V., Esposito, F., de Angelis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+endonasal+surgery+for+pituitary+adenomas.">Endoscopic endonasal surgery for pituitary adenomas.</a> World neurosurg. 82 (6 Suppl), S3-S11 (2014).</li><li>Aplin, J., Curtis, A. S. G., Lackie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+Matrices+for+Studing+Cell+Adhesion.">Model Matrices for Studing Cell Adhesion.</a> Measuring cell adhesion. , 110-111 (1991).</li><li>Guiraud, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prolactin-producing+pituitary+adenomas+in+three-dimensional+culture.">Human prolactin-producing pituitary adenomas in three-dimensional culture.</a> In. Vitro. Cell. Dev. Biol. 27 (3), 188-190 (1991).</li><li>Ma, H. L., Hung, S. C., Lin, S. Y., Chen, Y. L., Lo, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+human+mesenchymal+stem+cells+encapsulated+in+alginate+beads.">Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads.</a> J. Biomed. Mater. Res. A. 64 (2), 273-281 (2003).</li></ol>

The authors have nothing to disclose.

该协议有几个关键步骤。第一是珠粒度均匀性，这是需要保持营养物和气体的扩散的相同的条件下在所有的细胞培养物。根据我们的经验，采用了21g的针头，使藻酸盐可用于收购珠均匀大小的有效文化。第二个重要因素是从针的距离;这个距离必须不超过从钙溶液5cm至避免变形珍珠和死细胞由于与钙溶液的表面上的藻酸盐/细胞溶液流的碰撞。第三关键步骤是在玻璃搅拌在其中珠被丢弃，以防止它?...

三维支架已在细胞培养被广泛使用，因为它们提供用于培养细胞的三维结构支撑和细胞-细胞通讯1的维护。天然衍生的聚合物材料已用于制造三维支架，包括I型胶原蛋白，壳聚糖和藻酸盐。海藻酸钠是从褐色海藻Macrocystispyrifera（海带）的天然聚合物。该聚合物是由β-D-甘露糖醛酸（M）和α-L-古洛糖醛酸（G）的2和形式在某些二价阳离子，如钙和钡的存在下是稳定的凝胶的重复单元。氯化钙（ 氯化钙 ）溶液通常用作交联剂以形成藻酸盐珠。海藻酸钠溶液滴入氯化钙立即形成三维球凝胶。当藻酸盐溶液与细胞混合，将细胞封装在藻酸盐bEADS 3。 藻酸盐具有已启用它的属性被用作用于多种细胞，包括软骨4，骨骼肌成肌细胞5和神经干细胞6的包封的基质。它是一种惰性材料，并允许营养物，氧气和维持细胞的存活和功能的代谢产物的扩散;此外，不同于其他基于凝胶的培养系统，藻酸盐珠内培养的细胞，也可以从该支架使用钙螯合剂，如柠檬酸钠解放，然后将细胞可以收获进一步调查7。 垂体腺瘤通常是良性肿瘤低增殖率。大鼠垂体腺瘤细胞系已经在二维系统 8成功培养。然而，这种文化分泌人垂体细胞瘤是不是一个高效的开发;分散垂体肿瘤细胞生长不佳在培养中，细胞呈现出有限的附着和铺展，并将细胞通常形成在培养皿9,10-漂浮聚集。 已进行了多种尝试，以获得肿瘤垂体细胞悬浮液，包括酶和机械分散方法11，一个单独机械分散方法12和肿瘤外植体10的培养。用这些方法中，不同的作者获得可行的粘附培养不同的时间段。肿瘤分泌细胞的能力在这些两维系统生存取决于肿瘤的类型和它们的增殖率9。但是，在长期培养，与成纤维细胞表型细胞为主11,13。本文介绍了从封装在藻酸盐珠垂体腺瘤细胞进一步释放他们的海藻酸钠支架，能够进行详细的分析获得原代培养的方法它们的细胞生物学，例如，其细胞骨架的安排的各个方面。

<table><tbody><tr><td>&lt;strong&gt;Reagent&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>199 culture medium&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>31100-027</td><td>For culture, warm in a 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA</td><td>A15-751</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>106329</td><td></td></tr><tr><td>N-(2-Hydroxyethyl) piperazine-N&amp;acute;-2ethanesulfonic acid (HEPES)</td><td>Sigma&amp;nbsp;</td><td>H-4034</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Invitrogen&amp;nbsp;</td><td>21600-044</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>27250-018</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Research Organics</td><td>3002E</td><td></td></tr><tr><td>Sorbean trypsin inhibitor&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>17075-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>2139</td><td></td></tr><tr><td>Alginic acid, From Macrocystis Pyrifera (Kelp)</td><td>Sigma&amp;nbsp;</td><td>A-2158</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>J.T. Baker</td><td>1332</td><td></td></tr><tr><td>Sodium citrate (Na3C6H5O7)</td><td>J.T. Baker</td><td>3646</td><td></td></tr><tr><td>Piperazine-N,N&amp;prime;-bis(2-ethanesulfonic acid) (PIPES)</td><td>Research Organics</td><td>9624P</td><td></td></tr><tr><td>Polyethylene Glycol 6000 (PEG 6000)</td><td>Calbiochem</td><td>528877</td><td></td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>Research Organics</td><td>9574E</td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>2500</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Formaldehyde</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>J.T. Baker</td><td>660</td><td></td></tr><tr><td>BSA Bovine Serum Albumin IgG-Free</td><td>Jackson Immuno Research</td><td>001-000-161</td><td></td></tr><tr><td>Phalloidin&amp;ndash;Tetramethylrhodamine B isothiocyanate</td><td>Sigma</td><td>P1951</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>Invitrogen&amp;nbsp;</td><td>D1306</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>9681</td><td>Highly corrosive. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sodium hydroxide (NAOH)</td><td>Merck</td><td>1.06498</td><td>Can cause eye and skin irritation.Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>(3-Aminopropyl)triethoxysilane (APTS)</td><td>Sigma</td><td>A-3648</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Toxic: May cause cancer. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Rotator&amp;nbsp;</td><td>Boekel Scientific</td><td>Model 230300</td><td></td></tr><tr><td>Centrifuge</td><td>DuPont Corporation&amp;nbsp;</td><td>Model sorvall TC6</td><td></td></tr><tr><td>shaker&amp;nbsp;</td><td>Lab-line Instruments</td><td>Model 314-820&amp;nbsp;</td><td></td></tr></tbody></table>

three-dimensional alginate-bead culture of human pituitary adenoma cells

三维培养方法进行说明，其中主垂体腺瘤细胞生长在藻酸盐珠。海藻酸钠是从棕色海藻衍生的聚合物。简言之，将肿瘤组织切成小块并用胶原酶和胰蛋白酶提交酶促消化。接着，获得的细胞悬浮液。肿瘤细胞悬浮液与1.2％的藻酸钠混合，并落入氯化钙溶液，和藻酸盐/细胞悬浮液凝胶化上与氯化钙 2接触，以形成球形珠。嵌在藻酸盐珠将细胞用由有20％FBS富集培养基中提供营养物供给。在藻酸盐珠的三维培养保持腺瘤细胞的活力为长时间，最多四个月。此外，细胞可以从藻酸盐通过洗涤柠檬酸钠珠粒中释放并接种于玻璃盖玻片上用于进一步免疫细胞化学分析。使用CEL的升培养模式可以使用最少的解体细胞骨架的固定和可视化。总之，藻酸盐珠用于维修垂体腺瘤细胞的可靠培养系统。

该协议已经在培养，并在不同的时间段藻酸盐珠腺瘤垂体细胞的维持成功地应用于图1示出在藻酸盐珠包埋的细胞后三个月。这些细胞表现出的双折射的圆形形状的倒置光学显微镜（图1）下，图2示出了N-钙粘蛋白的嵌入在藻酸盐大鼠垂体腺瘤细胞中的定位。的N-钙粘蛋白在细胞-细胞接触（图2A 和 2C）的本地化?...

Watch this Scientific Journal Video about Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells at JoVE.com

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

three-dimensional-alginate-bead-culture-human-pituitary-adenoma

Research

JoVE Journal

Medicine

9.9K 次观看.  Center for Research and Advanced Studies (CINVESTAV-IPN). Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

视频: Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

科研

生物工程

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

癌症研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

医学

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Source: Koss, K. M, et al. <a href="https://app.jove.com/v/56615">Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to establish a three-dimensional culture of glial cells. Rat-brain-derived glial cells are encapsulated within a photocrosslinkable polymer matrix containing extracellular matrix components. Upon incubating in a growth medium, the glial cells proliferate within the supporting matrix to create a three-dimensional culture.

建立大鼠脑源性神经胶质细胞的三维培养物

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...